Individual Platelet Adhesion Assay: Measuring Platelet Function and Antiplatelet Therapies in Whole Blood via Digital Quantification of Cell Adhesion

Background The hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis. Age-dependent values of pro- and anti-coagulant proteins have been determined. However, the task of defining age-dependent normal values of neonatal platelet function has been met with challenges owing to difficulties in obtaining adequate blood volumes for functional assays and inconsistent results amongst varying testing methods. In order to overcome many of these challenges, cord blood is often used as a source of neonatal platelets. Platelet aggregometry comparing adult and cord blood derived platelets has demonstrated a near lack of platelet response to epinephrine, collagen, and thromboxane in cord blood samples. In contrast, other studies of platelet function, such as flow cytometry, have failed to demonstrate this phenotypic difference. Assays of primary hemostasis reveal that neonatal blood mediates primary hemostasis as effectively as adult blood. In order to overcome the challenges associated with studying neonatal platelets, we have developed a novel platelet function assay employing small volumes of blood obtained directly from the neonate in order to assess platelet adhesion, activation, and aggregation simultaneously. Methods Eight-well slide chambers were coated with either fibrillar collagen or fibrinogen and allowed to adsorb at room temperature for one hour. Blood was obtained from healthy adult controls via venipuncture and neonatal samples via heelstick into sodium citrate. The blood was separated into two 200 µl aliquots, and TRAP (Thrombin Receptor Activating Peptide: 30 mM) was added to one aliquot. 100 µl of plain whole blood was added to both a collagen and a fibrinogen coated well and 100 µ of whole blood plus TRAP was added to a fibrinogen coated well. The samples were then incubated at 37°C for 30 minutes. Non-adherent cells were washed three times with modified HEPES-Tyrode buffer. FITC-P-selectin was then added (10 µg/ml), and the samples were incubated at 37 oC for 10 minutes and subsequently washed. Samples were imaged with differential interference contrast (DIC) and fluorescence microscopy on a Zeiss Axiovert 200 M microscope. Results Platelet adhesion, activation, and aggregation were assessed for 3 neonatal samples and 3 adult control samples. Both adult and neonatal platelets adhered to fibrinogen and collagen equally. Exposure to collagen and fibrinogen (+/- TRAP) resulted in alpha granule release and P-selectin expression in both neonatal and adult platelets. In addition, both adult and neonatal platelets were observed to undergo the characteristic cytoskeletal changes that result in platelet spreading on fibrinogen (+/- TRAP) and collagen surfaces. Both neonatal and adult platelets were observed to form platelet aggregates on both surfaces under static conditions. (Figure 1) Conclusions We have successfully developed a novel platelet function assay using small volumes of whole blood to assess three key platelet functions: adhesion, activation, and aggregation. This is the first study to demonstrate that neonatal platelets spread on adhesive and extracellular matrix proteins and suggests that neonatal platelets contain the cytoskeletal machinery necessary to undergo this change in platelet formation. This assay fills a critical need in clinical pediatric hematology where efforts to diagnose and treat neonatal platelet dysfunction are often met with technical challenges related to conventional platelet function assays. Disclosures: No relevant conflicts of interest to declare.

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Pentamidine Is a Specific, Non-Peptide, GPIIb/llla Antagonist

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650305 ◽

1996 ◽

Vol 75 (03) ◽

pp. 503-509 ◽

Cited By ~ 12

Author(s):

Dermot Cox ◽

Toshiaki Aoki ◽

Jiro Seki ◽

Yukio Motoyama ◽

Keizo Yoshida

Keyword(s):

Monoclonal Antibody ◽

Cell Adhesion ◽

Endothelial Cell ◽

Intracellular Ph ◽

Platelet Adhesion ◽

Adhesion Assay ◽

Agonist Peptide ◽

A Cell ◽

Granule Release ◽

Endothelial Cell Adhesion

SummaryPentamidine was previously shown to act on glycoprotein (GP) Ilb/IIIa (Cox et al., Thromb Haemost 1992; 68: 731). In this paper we study the effect of pentamidine on other RGD-dependent receptors. In a cell adhesion assay, pentamidine was 500 times more potent than RGDS at inhibiting platelet adhesion to fibrinogen. While RGDS inhibited platelet adhesion to fibronectin, endothelial cell adhesion to vitronectin or fibronectin, 293 cell adhesion to vitronectin, IMR 32 cell adhesion to fibronectin and C32 cell adhesion to vitronectin; pentamidine failed to inhibit these interactions at doses as high as 1 mM. Resting platelets fixed in the presence of 1 mM RGDS had increased binding of fibrinogen, i.e., RGDS activated GPIMIIa, while pentamidine at 100 ΜM had no effect. Similarly, RGDS induced the binding of an anti-LIBS monoclonal antibody, while pentamidine had no effect. Pentamidine partially, but significantly, inhibited lysosome and a-granule release induced by the thrombin agonist peptide, while RGDS had no effect. Neither pentamidine nor RGDS affected ADP-induced Ca2+ influx. Pentamidine had no effect on ADP-induced intracellular pH changes while RGDS prevented the pH from returning to normal. Thus, pentamidine is a non-peptide GPIIb/IIIa antagonist that is non-activating and is specific for GPIIb/IIIa.

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Fibrinogen Adsorption, Platelet Adhesion and Thrombin Generation at Heparinized Surfaces Exposed to Flowing Blood

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613074 ◽

2002 ◽

Vol 87 (04) ◽

pp. 742-747 ◽

Cited By ~ 26

Author(s):

George Willems ◽

Marco Morra ◽

Jeffrey Keuren ◽

Simone Wielders ◽

Theo Lindhout

Keyword(s):

Protein Adsorption ◽

Whole Blood ◽

Thrombin Generation ◽

Platelet Adhesion ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Adhesion Assay ◽

Flowing Blood ◽

Fibrinogen Adsorption

SummaryThrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anticoagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.

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Familial Antithrombin III Deficiency Associated With Recurrent Arterial Thromboembolism

10.1055/s-0038-1652850 ◽

1981 ◽

Author(s):

C S Hale ◽

J C Mattson ◽

J A Zuhlke

Keyword(s):

Platelet Aggregation ◽

Family History ◽

Platelet Function ◽

Platelet Adhesion ◽

Antithrombin Iii ◽

Family Members ◽

Adhesion Assay ◽

Arterial Thromboembolism ◽

At Iii ◽

History Of

A 28-year-old male with a strong family history of thromboembolic disease sustained three arterial thromboembolic occlusions during a 7 mo. period. The patient had antithrombin III (AT III) levels of 19.2 mg/dl (N=17-30 mg/dl) by RID and an immediate (heparin activated) antithrombin level of 77% (N=88-120%). Crossed IEP showed normal electrophoretic mobility of the patient's AT III. Of 30 family members tested, 10 demonstrated decreased AT III by both immunologic and functional assays. Two children of the propositus and three children of the sister of the propositus were tested and none were found to be abnormal. Because of the unusual presence of arterial thrombotic events in this family, platelet function studies were performed on the propositus and on two AT III deficient family members. A 19-year-old brother of the propositus, with no history of thrombotic events but with 13 mg/dl AT III and 83% activity had normal platelet aggregation studies and demonstrated no evidence of hyperaggregability with suboptimal concentrations of aggregating agents. He did, however, demonstrate a slight increase in platelet adhesion in a collagen adhesion assay. The second family member, a 65 year-old aunt of the propositus with 13.7 mg/dl AT III and 75% activity was on Coumadin at the time of evaluation of platelet function. Platelet aggregation and adhesion studies were normal in this individual. The propositus was also tested while on Coumadin and showed no abnormality of platelet aggregation, no hyperaggregability and no increase in platelet adhesion.In view of our experience, we recommend screening for AT III deficiency in patients with unexplained recurrent arterial thromboembolism as well as those with venous thromboembolism, especially if the family history is suggestive.

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Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC)

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00385.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. G167-G176 ◽

Cited By ~ 26

Author(s):

Parvaneh Rafiee ◽

Daniel J. Stein ◽

Victoria M. Nelson ◽

Mary F. Otterson ◽

Reza Shaker ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Whole Blood ◽

Cell Adhesion Molecule ◽

Microvascular Endothelial Cells ◽

Adhesion Assay ◽

Tnf Α ◽

Cox 2 ◽

Microvascular Endothelial

The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-α but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-α/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-α/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-κB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-α/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-κB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD.

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Fine Structure of Platelet Interaction with a New Microcrystalline Collagen Preparation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050305 ◽

1974 ◽

Vol 32 ◽

pp. 58-59

Author(s):

W. H. Zucker ◽

R. G. Mason

Keyword(s):

Fine Structure ◽

Platelet Aggregation ◽

Hydrochloric Acid ◽

Whole Blood ◽

Platelet Adhesion ◽

Hemostatic Agent ◽

Native Collagen ◽

Fibrillar Morphology ◽

Clinical Interest ◽

New Form

Platelet adhesion initiates platelet aggregation and is an important component of the hemostatic process. Since the development of a new form of collagen as a topical hemostatic agent is of both basic and clinical interest, an ultrastructural and hematologic study of the interaction of platelets with the microcrystalline collagen preparation was undertaken.In this study, whole blood anticoagulated with EDTA was used in order to inhibit aggregation and permit study of platelet adhesion to collagen as an isolated event. The microcrystalline collagen was prepared from bovine dermal corium; milling was with sharp blades. The preparation consists of partial hydrochloric acid amine collagen salts and retains much of the fibrillar morphology of native collagen.

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Cell adhesion assay to determine the interaction between NGL-3 with LAR cell adhesion molecules

Protocol Exchange ◽

10.1038/nprot.2009.71 ◽

2009 ◽

Author(s):

Jooyeon Woo ◽

Seok-kyu Kwon ◽

Eunjoon Kim

Keyword(s):

Cell Adhesion ◽

Adhesion Molecules ◽

Cell Adhesion Molecules ◽

Adhesion Assay ◽

Cell Adhesion Assay

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Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646346 ◽

1992 ◽

Vol 68 (06) ◽

pp. 694-700 ◽

Cited By ~ 25

Author(s):

Roy R Hantgan ◽

Silvia C Endenburg ◽

I Cavero ◽

Gérard Marguerie ◽

André Uzan ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Platelet Adhesion ◽

Integrin Receptor ◽

Shear Rates ◽

Perfusion Chamber ◽

Receptor Recognition ◽

Adhesive Interactions ◽

Coated Surfaces ◽

Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

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Evaluation of Stemness Maintenance Properties of the Recombinant Human Laminin α2 LG1-3 Domains in Human Mesenchymal Stem Cells

Protein and Peptide Letters ◽

10.2174/0929866526666190617091155 ◽

2019 ◽

Vol 26 (10) ◽

pp. 785-791

Author(s):

Ji-Eun Kim ◽

Hye-Jin Seo ◽

SuJin Lee ◽

Jun-Hyeog Jang

Keyword(s):

Escherichia Coli ◽

Stem Cells ◽

Molecular Weight ◽

Mesenchymal Stem Cells ◽

Cell Adhesion ◽

Human Mesenchymal Stem Cells ◽

Adhesion Assay ◽

Proliferation Assay ◽

Undifferentiated State ◽

Laminin Α2

Background: Laminin, a member of the Extracellular Matrix (ECM), is a glycoprotein that is used as a factor that affects cell adhesion, proliferation, survival, and differentiation. Of these, five globular domains (LG domains) of the alpha chain play an important role in influencing the cell by binding to the integrin. Objective: This study aimed to evaluate the ability of globular domains 1-3 of laminin alpha2 (rhLAMA2LG1-3) in maintaining the pluripotency of human Mesenchymal Stem Cells (hMSCs), which are widely used in regenerative medicine. Methods: hMSCs were grown in the medium supplemented with rhLAMA2LG1-3, then the effect of the protein on hMSCs were confirmed through cell adhesion assay, proliferation assay and RTPCR. Results: rhLAMA2LG1-3 expressed in Escherichia coli has a molecular weight of 70 kDa, at 1 µg/ml concentration of rhLAMA2LG1-3, the attachment and proliferation of hMSCs were approximately 3.18-fold and 1.67-fold, respectively, more efficient than those of untreated controls. In addition, the undifferentiated state and degree of stemness of hMSCs were measured, on the basis of CD90 and CD105 levels. In the rhLAMA2LG1-3-treated hMSCs, the expression levels of CD90 and CD105 increased by 2.83-fold and 1.62-fold, respectively, compared to those in untreated controls. Conclusion: rhLAMA2LG1-3 can be potentially used in stem cell therapy to improve the viability and maintain the undifferentiated state of hMSCs.

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